We want to do some FACS analysis on freshly isolated primary mouse hepatocytes. So far, we had problems with high autofluorescence. Any suggestions to solve this problem? Can the isolation method be the problem?
Thanks for advice.
Autofluorescene is caused by the cells natural fluorescence, mostly cyclic ring compounds including NAD(P)H, collagen, riboflavin as well as the aromatic amino acids. Most of these absorb light in the UV and blue laser ranges (355 to 480nm) and emit in the green range (350 - 550nm) unfortunately its a consequence of the cell type you are working on and it will mean your signal sensitivity decreases in these channels. I get large autofluorescence with my dendritic cells are the larger the cells the more of these compounds they have the more autofluorescent they will be. As I understand hepatocytes are pretty much powerhouses for protein synthesis and other metabolic processes so they will be high in all of these molecules!
Try to avoid using antibodies in the green range if you can, my most sensitive markers I put in the red / far red wavelengths to try and avoid this but thats not always possible. First step to do is to Fc Block which is always important with high autofluorescent cells, then titrated your antibodies if you haven't already this will help you figure out the best signal to noise ratio for your samples.
Hello Christian,
I had good experience by gating out the dead hepatocytes, e.g. with PI.
Greetings
C. Leder
Sorry Christian I also forgot to mention make sure you are using FMO controls on these cells if you are doing mutlicolour flow cytometry these are incredibly useful for checking to see which channels the autofluorescene is bleeding into and to what extent the antibodies are spilling over. Also I'm sure I'm preching to the choir here but you must have single stain controls that are brighter than your cells for compensation.
Try to adjust color compensation using unstained hepatocytes. Also, Fc Blocking did make some difference for us... Analysing hepatocytes with FACS is always challenging. Good luck Christian.
we did diskriminate gfp+ cells with high autofluorescene. so we made a plot fl1 vs. fl2
and so you can see verry fine 3 populations (importent make no compensation). see atachment
So you can discrimeanate autoflourecente
Hi,
Could any one please tell me which surface markers one should use for hepatocytes?
thank you
And would anyone please have an example of how a whole liver cell suspension (all cell types in there) looks like in the FSC/SSC, and if the distinct populations (e.g. hepatocytes, edothelial cells and kupffer cells) can be clearly distinguished?
We tried to look at a mouse liver cell suspension for the first time but we do not see any clearly defined populations. We also failed to see clear autofluorescence of the hepatocytes.
Dear Christian
Which marker do you use for flow cytometry of hepatocytes.
Thanks
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